The present invention relates to methods and compositions for enhancing an effect of a neutrophin, preferably, but not limited to enhancing the growth and survival promoting properties of neurotrophins, using an analogue of a portion of p75NTR.
The neurotrophins are a family of structurally and functionally related neurotrophic factors. The family includes prototypic member nerve growth factor (NGF), as well as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) (Heumann, 1994) and neurotrophin-6 (NT-6) (Gotz et al., 1994). The neurotrophins have similar structural conformations, including three surface xcex2-hairpin loops, a xcex2-strand, an internal reverse turn region, and N- and C-termini, and exhibit approximately 50% amino acid sequence identity.
Neurotrophins function to promote growth and survival of certain classes of peripheral and central nervous both during development and following neuronal damage. For example, NGF is involved in the development of neurons in the peripheral nervous system, supports neuronal survival, and enhances and maintains the differentiated state of neurons. Neurotrophins can promote neurite differentiation such as sprouting or process formation, and process growth. Neurotrophins can also modulate cell motility (Anton et al., 1994), for example, both accelerate nerve process growth and decrease general cell motility. Another neurotrophin-mediated activity is induction of particular enzymes.
Furthermore, with respect to functional similarity, each of the neurotrophins can bind to a membrane-bound receptor protein (MWxcx9c75 kDa) called the common neurotrophin receptor, or xe2x80x9cp75NTRxe2x80x9d. Each neurotrophin also binds with higher affinity to a second membrane-bound receptor protein of the tyrosine kinase receptor (Trk) family. In particular, NGF binds selectively to the TrkA receptor, and BDNF and NT-4/5 bind selectively to the TrkB receptor. NT-3 is less selective and, though it binds primarily to the TrkC receptor, NT-3 also exhibits some binding to the TrkA and TrkB receptors (banez et al., 1993).
A variety of cell types express either p75NTR and/or a member of the Trk family of receptor tyrosine kinases. These include neurons, mast cells, glial cells such as astrocytes, oligodendrocytes and Schwann cells, and dysplasic or malignant cells such as neuroblastoma or melanoma cells. Cells of neuronal lineage that differentiate by extension of neurites in the presence of a neurotrophin express both a member of the Trk receptor family and lower molecular weight receptor protein p75NTR.
Neurite growth is the best characterized differentiation response to NGF, and evidence is beginning to emerge that p75NTR can modulate this activity. Gene targeting studies resulting in nonfunctional p75NTR demonstrate reduced density of sensory and sympathetic innervation in vivo (Lee et al., 1994 a and b), possibly related to a shift to the right of dose response curves for NGF (Davies et al., 1993). The low molecular weight neurotrophin receptor p75NTR is a member of a family of receptors designated the NGF receptor superfamily (Krammer and Debatin, 1992; Mallett and Barclay, 1991). In addition to p75NTR, this family includes TNFR1, TNRF2, CD30, Fas, Fas/Apo-1, Apo-3, CD40, 4-1BB, CD-27, SFV-T2, and OX-40. While these receptors have been grouped according to structural similarities in transmembrane and extracellular domains, the inventors have provided evidence supporting existence of shared pulative signalling motifs in the cytoplasmic domains (Myers et al., 1994, incorporated herein by reference). Similar to the wasp venom tetradecapeptide mastoparan (MP), p75NTR (rat, chick, human), human TNFR-1, and human 4-1BB have been found to have secondary structure domains with putative plasma membrane associating properties that have been implicated in intracellular signalling (Higashijima, 1983, 1990, each incorporated herein by reference).
Fas/Apo-1, TNFR1, Apo-3, and p75NTR all activate apoptosis via xe2x80x9cdeath domainsxe2x80x9d that are rich in sequences predicted to form xcex1-helices (Myers et al., 1994). These xcex1-helices are potentially involved in oligomerization that mediates signalling in ligand-independent or ligand-dependent states (Huang et al., 1996). TNFR1 and p75NTR contain within their death domain regions, motifs that confer surface membrane associating properties (Myers et al., 1994) as determined by maximum mean hydrophobic moment (Eisenberg et al., 1984a,b), and both TNFR1 and p75NTR signal translocation of transcription factor NFkB (Marsters et al., 1996; Carter et al., 1996). In contrast, Apo-1/Fas, which contains a death domain but no amphiphilic sequence therein (Myers et al., 1994), does not translocate NFkB (Marsters et al., 1996). The inventors reasoned from these observations that the amphiphilic motif of p75NTR could be involved in NFkB activation/translocation, and thus conducted a series of experiments to determine whether the amphiphilic motif of p75NTR and NFkB interact directly.
Based on knowledge of their biological actions, neurotrophins have been considered for therapeutic use in neurological disorders characterized by loss of neurons and/or loss of connectivity. Such disorders include stroke, cerebral and spinal cord injury, a host of neurodegenerative disorders, the most prevalent of which is Alzheimer""s disease, and a variety of peripheral nerve disorders, most notably those associated with diabetes mellitus and cancer chemotherapy.
These are limitations to use of neurotrophins for therapy of neurological disorders. Such limitations relate to the prohibitive costs of production of recombinant human neurotrophins, their stability in vivo, the routes of administration, and penetration of the blood-brain and nerve-brain barriers.
To circumvent such limitations, stable small molecule neurotrophin agonists or stable small molecule modulators of neurotrophin levels or neurotrophin effects would be desirable.
It is an object of the present invention to provide methods and compositions for enhancing an effect of a neurotrophin, preferably enhancing the growth and survival promoting properties of a neurotrophin.
It is a further object to provide low molecular weight analogues of p75NTR 367-379 useful as therapeutic agents for in situ treatment of certain neurological disorders.
It is a yet another object to provide low molecular weight analogues of p75NTR 367-379 useful for ex vivo treatment of cells. Such ex vivo uses include diagnostic applications, assays wherein various factors are screened, and applications wherein cells, e.g., hematopoientic cells, are treated prior to their reintroduction to a patient.
Utilizing peptide analogues of a putative amphiphilic domain of p75NTR, the inventors have discovered that these analogues have biophysical and biochemical properties in MP (Sequence ID No. 5) that they are taken up into cells, that the amphiphilic properties and polar group relationships of p75NTR 367-379 analogues influence signalling pathways involved in neurite growth by NGF-responsive cells of neuronal lineage, and that these analogues depend upon the functional expression of TrKA to exert their influence. The findings suggest that p75NTR participates by interaction of its amphiphilic domain with a Trk receptor to modulate neurite growth.
In the broadest aspect, the present invention provides a method for enhancing the effect of a neurotrophin on cells having a plasma membrane and expressing a neurotrophin receptor. The method comprises providing an effective compound having an effective number of charged moieties spaced thereon and, when in close proximity to the plasma membrane, an amphipathic conformation comprising a substantially hydrophobic membrane associating face and a substantially hydrophilic face. The method includes exposing the cells for an effective period of time to an effective amount of the compound in the presence of the neurotrophin for enhancing the effect of the neurotrophin on the cells. In a first preferred embodiment, the compound is a peptide or peptidomimetic. In a second preferred embodiment, the cells are of a neuronal lineage, and the effect is growth and/or survival. In a third preferred embodiment, the neurotrophin is nerve growth factor (NGF) and the receptor is TrkA.
In this aspect of the invention, the effective compound is an analogue of an effective putative amphiphilic domain of p75NTR, and the neurotrophin receptor is selected from the nerve growth factor receptor superfamily.
In some embodiments, the analogue is a peptide chain comprising amino acid residues and having an amphipathic portion. The amphipathic portion includes a substantially hydrophobic membrane associating face and a substantially hydrophilic face. Further, in certain embodiments, the amphipathic portion is an xcex1-helix having a periodicity in hydrophobicity of the amino acid residues effective to form the substantially hydrophobic membrane associating face and the substantially hydrophilic face. The xcex1-helix may comprise a sequence of about eleven amino acid residues. In other embodiments, the peptide chain may have substantially hydrophobic membrane associating face and a substantially hydrophilic face that are couple by a turn. At least one of the faces may be a xcex2-strand or other organized secondary structure; for example, the membrane associating substantially hydrophobic face may be a xcex2-strand.
A preferred peptide analogue in this aspect of the invention comprises a peptide sequence LDALLAALRRIQR (Sequence ID No. 1) and the neurotrophin receptor is tyrosine kinase family member Trk A.
In other embodiments of this aspect of the invention, the effective compound is not a peptide, but mimics amphipathic properties and functional effects of p75NTR 367-379.
The invention provides a method for enhancing the effect of a neurotrophin on cells having a membrane, a DNA binding protein in their cytoplasm, and expressing a neurotrophin receptor. The method comprises providing an effective compound, the compound having an effective number of charged moieties spaced thereon and, when in close proximity to the membrane a conformation comprising a membrane associating substantially hydrophobic face and a substantially hydrophilic face for associating with the DNA binding protein. The method includes exposing the cells for an effective period of time to an effective amount of the compound in the presence of a neurotrophin for enhancing growth or survival of the cells.
In another aspect of the invention there is provided an amphipathic compound for enhancing an effect of a neurotrophin on cells having a membrane and expressing a neurotrophin receptor. The compound comprises a substantially hydrophobic portion for associating with the membrane and a substantially hydrophilic portion comprising charged moities, polar moities or combinations thereof for interacting with a factor partaking in a mechanism of the enhancing effect including but not limited to nuclear transcription factors.
The present invention provides an amphipathic peptidomimetic compound for enhancing an effect of a neurotrophin on cells having a membrane and expressing a neurotrophin receptor. The compound comprises a substantially hydrophobic portion of associating with the membrane and a substantially hydrophilic portion comprising charged moieties, polar moieties or a combination of both for interacting with a factor partaking in a mechanism of the enhancing effect.
In another aspect of the invention there is provided a method for enhancing neurotrophin-mediated growth and/or survival of cells having a plasma membrane and expressing a neurotrophin receptor. Preferably, the cells are of a neuronal lineage. The method comprises exposing the cells for an effective period of time to an effective compound.
The peptide compound is a peptide analogue of an effective putative amphiphilic domain of p75NTR and the peptide analogue is a linear peptide chain comprising a sequence of at least about level amino acid residues. The peptide compound may have a periodicity in hydrophobicity of the amino acid residues effective to form a membrane associating amphipathic xcex1-helix portion in close proximity to the plasma membrane. The xcex1-helix portion has a substantially hydrophobic face and a substantially hydrophilic face with the compound having an effective number of charged moieties spaced thereon. In an alternate preferred embodiment, other organized secondary structural motifs cooperate to form a substantially hydrophobic membrane associating domain and a substantially hydrophilic domain.
The present invention provides a method for enhancing an effect of a neurotrophin on cells having a plasma membrane and expressing a neurotrophin receptor. The cells may be of a neuronal lineage and the effect may be nerve growth factor-mediated neurite growth. The method comprises exposing the cells for an effective period of time to a medium comprising an effective amount of a peptide having a membrane associating amphipathic xcex1-helix in close proximity to said plasma membrane. The peptide may have a sequence LDALLAALRRIQR (Sequence ID No. 1).
In another aspect the invention provides a method for enhancing an effect of a neurotrophin, preferably nerve growth factor-mediated neurite growth in cells of a neuronal lineage. The method comprises providing cells of a neuronal lineage having a surface membrane and being capable of expressing a neurotrophin receptor. The method includes exposing the cells for an effective period of time to a medium comprising an effective amount of nerve growth factor and a peptide having a sequence LDALLAALRRIQR (Sequence ID No. 1) wherein the sequence forms a membrane associating amphipathic xcex1-helix in close proximity to the surface membrane.
In this aspect of the invention the neurotrophin receptor is selected from the tyrosine kinase family and more particularly is tyrosine kinase family member TrkA.
The present invention provides a use for an effective compound for enhancing an effect of a neurotrophin in cells having a plasma membrane and expressing a neurotrophin receptor. Preferably, the cells are of a neuronal lineage and the effect is nerve growth factor-mediated neurite growth. The use for the effective compound comprises providing a medium comprising the effective compound, wherein the compound is an analogue of an effective putative amphiphilic domain of p75NTR. The cells are exposed to an effective amount of the compound for an effective period of time in the presence of a neurotrophin for enhancing neurite growth in the cells. The analogue may be a linear peptide chain comprising a sequence of about eleven amino acid residues having an effective number of charged moieties spaced thereon and having a periodicity in hydrophobicity of the amino acid residues so that said compound has a membrane associating amphipathic xcex1-helix portion in close proximity to said plasma membrane. The xcex1-helix portion has a substantially hydrophobic face and a substantially hydrophilic face.
In this aspect of the method the peptide analogue may have an amino acide sequence LDALLAALRRIQR (SEQ. ID NO.: 1) or a functional equivalent thereof.